ABSTRACT
In a previous study using a microRNA (miRNA/miR) microarray assay, we demonstrated that miR-133b-5p was upregulated in response to hypoxic preconditioning (HPC). The present study was designed to investigate the role of the miR133b5p in HPCinduced cardioprotection and the underlying mechanisms involving caspase8 and caspase3 apoptotic signaling. Adult rats were subjected to myocardial ischemia/reperfusion (I/R) injury with or without ischemic preconditioning (IPC), and the level of miR133b5p in myocardium was measured. Neonatal rat cardiomyocytes were isolated and subjected to hypoxia/reoxygenation (H/R) injury, with or without HPC. miR133b5p antagomir was transfected into the cardiomyocytes to observe whether it could block HPCinduced cardioprotection. Cellular injury was evaluated by detecting cell viability, lactate dehydrogenase (LDH) activity and apoptotic rate. Reverse transcriptionquantitative polymerase chain reaction was used to measure the level of miR133b5p. The activation of caspase8 and caspase3 were measured by western blot analysis to detect the cleaved fragments as well as a colorimetric assay. Following myocardial I/R injury, the expression of miR133b5p was decreased in myocardium, while this decrease was restored by IPC. HPC protected neonatal rat cardiomyocytes against H/R injury by increasing cell viability, while reducing LDH release and cell apoptosis. These protective effects were coupled with the upregulation of miR133b5p. However, the knockdown of miR133b5p in the cardiomyocytes blocked HPCmediated cardioprotection as reflected by the aggravation of cell injury and apoptosis. HPC upregulated miR133b5p level was markedly suppressed by the antagomir. In addition, the cleavage and activities of caspase8 and caspase3 were inhibited by HPC while reversed by knockdown of miR133b5p. Upregulation of miR133b5p contributes to HPCmediated cardioprotection in cardiomyocytes, and the mechanism may be associated with inhibition of caspase8 and caspase3 apoptotic signaling.
Subject(s)
Caspase 3/metabolism , Caspase 8/metabolism , Enzyme Activation , Ischemic Preconditioning, Myocardial , MicroRNAs/genetics , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/therapy , Animals , Apoptosis , Cells, Cultured , MicroRNAs/metabolism , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Rats, Sprague-Dawley , Up-RegulationABSTRACT
AIMS: Ischemia reperfusion (I/R) injury is an inevitable event arising during the cardiovascular diseases development and the process of potent surgical treatments. microRNAs (miRNAs) are critical regulators of multiple cell processes including I/R injury. The present study aims to quantify miRNA alterations and regulated genes upon hypoxia-reoxygenation (H/R) injury in a rat heart failure model comparing with normal cardiomyocytes. MAIN METHODS: Chronic heart failure was established by injecting doxorubicin (2mg/kg/week) for 6weeks, then H/R was performed on primary cultured cardiomyocytes isolated from normal and failed heart. Cellular injury was evaluated by detecting LDH release levels, cell variability and apoptotic rate. Dysregulated miRNAs in control, hypoxia preconditioning (HPC) and morphine preconditioning (MPC) groups under two conditions were quantified by microarray analysis. Fas protein expression was analyzed using Western Blotting analysis. KEY FINDINGS: Chronic heart failure was confirmed with lower ejection fraction (EF), and significant cellular injury. HPC could reverse the injury induced by H/R in normal heart rather than failed heart, otherwise, MPC significantly attenuated cellular injury dose dependently in both conditions. There was 12 miRNAs significantly altered after doxorubicin injection, 7 downregulated and 5 upregulated. miR-133b-5p, miR-6216, miR-664-1-5p and let7e-5p were differentially expressed after HPC and MPC treatments. The direct interaction between miR-133b-5p and target gene Fas were established. The Fas protein expression was manipulated by MPC not HPC affording protective effect against H/R injury. SIGNIFICANCE: We investigated that miR-133b-5p might play a particularly important role in the cardioprotective effect of MPC by regulating the target gene Fas.
Subject(s)
Hypoxia , MicroRNAs/metabolism , Morphine/therapeutic use , Myocardial Reperfusion Injury/metabolism , fas Receptor/metabolism , Animals , Apoptosis/genetics , Cluster Analysis , Disease Models, Animal , Doxorubicin/chemistry , Gene Expression Profiling , Gene Expression Regulation , Heart Failure/metabolism , L-Lactate Dehydrogenase/metabolism , Male , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/prevention & control , Myocytes, Cardiac/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: MicroRNAs (miRNAs) have been implicated in ischemia-reperfusion injury and ischemic preconditioning. Opioid pre- and postconditioning have powerful protective effects on the heart, but it is still not known whether miRNAs are involved in opioid-induced cardioprotection. The present study was designed to investigate the role of miRNAs in morphine preconditioning (MPC)-induced cardioprotection. METHODS: MiRNA microarray analysis was performed to examine the differentially expressed miRNAs caused by MPC in adult rat cardiomyocytes. A dual-luciferase reporter assay was performed to confirm the direct regulation of miR-133b-5p on the target gene Fas. MiR-133b-5p mimic or inhibitor was separately transfected into myocardial H9c2 cells to examine the role of miR-133b-5p in morphine-induced cardioprotection. RESULTS: MPC protected adult rat cardiomyocytes against hypoxia/reoxygenation (H/R) injury by reducing cell injury and death. MiRNA microarray data showed that a total of 39 miRNAs were differentially expressed after MPC treatment. A Dual-luciferase reporter assay confirmed that miR-133b-5p directly targets the Fas gene. After H/R injury, the decrease in miR-133b-5p and a contemporaneous rise in Fas mRNA and protein levels in adult rat cardiomyocytes were prevented by MPC treatment. In H9c2 cardiomyocytes, overexpression of miR-133b-5p reduced H/R-induced cell injury and apoptosis by inhibiting Fas expression. Knockdown of miR-133b-5p blocked morphine-mediated cardioprotection by reducing miR-133b-5p levels while enhancing the expression of Fas mRNA and protein. CONCLUSIONS: MPC causes a change in miRNA expression in rat cardiomyocytes. Morphine may protect cardiomyocytes against H/R injury through upregulation of miR-133b-5p by targeting Fas.